1. Field of the Invention
The present invention relates to antigen binding molecules (ABMs). In particular embodiments, the present invention relates to recombinant monoclonal antibodies, including chimeric, primatized or humanized antibodies specific for human carcinoembyronic antigen (CEA). In addition, the present invention relates to nucleic acid molecules encoding such ABMs, and vectors and host cells comprising such nucleic acid molecules. The invention further relates to methods for producing the ABMs of the invention, and to methods of using these ABMs in treatment of disease. In addition, the present invention relates to ABMs with modified glycosylation having improved therapeutic properties, including antibodies with increased Fc receptor binding and increased effector function, such as ADCC.
2. Background Art
Carcinoembryonic Antigen (CEA) and Anti-CEA Antibodies
Carcinoembryonic antigen (CEA, also known as CEACAM-5 or CD66e) is a glycoprotein having a molecular weight of about 180 kDa. CEA is a member of the immunoglobulin superfamily and contains seven domains that are linked to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor (Thompson J. A., J Clin Lab Anal. 5:344-366, 1991) The seven domains include a single N-terminal Ig variable domain and six domains (A1-B1-A2-B2-A3-B3) homologous to the Ig constant domain (Hefta L J, et al., Cancer Res. 52:5647-5655, 1992).
The human CEA family contains 29 genes, of which 18 are expressed: 7 belonging to the CEA subgroup and 11 to the pregnancy-specific glycoprotein subgroup. Several CEA subgroup members are thought to possess cell adhesion properties. CEA is thought to have a role in innate immunity (Hammarström S., Semin Cancer Biol. 9(2):67-81 (1999)). Because of the existence of proteins closely related to CEA, it can be challenging to raise anti-CEA antibodies that are specific for CEA with minimal cross-reactivity to the other closely related proteins.
CEA has long been identified as a tumor-associated antigen (Gold and Freedman, J Exp Med., 121:439-462, 1965; Berinstein N. L., J Clin Oncol., 20:2197-2207, 2002). Originally classified as a protein expressed only in fetal tissue, CEA has now been identified in several normal adult tissues. These tissues are primarily epithelial in origin, including cells of the gastrointestinal, respiratory, and urogential tracts, and cells of colon, cervix, sweat glands, and prostate (Nap et al., Tumour Biol., 9(2-3):145-53, 1988; Nap et al., Cancer Res., 52(8):2329-23339, 1992).
Tumors of epithelial origin, as well as their metastases, contain CEA as a tumor associated antigen. While the presence of CEA itself does not indicate transformation to a cancerous cell, the distribution of CEA is indicative. In normal tissue, CEA is generally expressed on the apical surface of the cell (Hammarström S., Semin Cancer Biol. 9(2):67-81 (1999)), making it inaccessible to antibody in the blood stream. In contrast to normal tissue, CEA tends to be expressed over the entire surface of cancerous cells (Hammarström S., Semin Cancer Biol. 9(2):67-81 (1999)). This change of expression pattern makes CEA accessible to antibody binding in cancerous cells. In addition, CEA expression increases in cancerous cells. Furthermore, increased CEA expression promotes increased intercellular adhesions, which may lead to metastasis (Marshall J., Semin Oncol., 30(a Suppl. 8):30-6, 2003).
CEA is readily cleaved from the cell surface and shed into the blood stream from tumors, either directly or via the lymphatics. Because of this property, the level of serum CEA has been used as a clinical marker for diagnosis of cancers and screening for recurrence of cancers, particularly colorectal cancer (Goldenberg D M., The International Journal of Biological Markers, 7:183-188, 1992; Chau I., et al., J Clin Oncol., 22:1420-1429, 2004; Flamini et al., Clin Cancer Res; 12(23):6985-6988, 2006). This property also presents one of the challenges for using CEA as a target, since serum CEA binds most of the currently available anti-CEA antibodies, hindering them from reaching their target on the cell surface and limiting potential clinical effects.
Multiple monoclonal antibodies have been raised against CEA for research purposes, as diagnostic tools, and for therapeutic purposes (e.g., Nap et al., Cancer Res., 52(8):2329-23339, 1992; Sheahan et al., Am. J. Clin. Path. 94:157-164, 1990; Sakurai et al., J. Surg. Oncol., 42:39-46, 1989; Goldenberg D M., The International Journal of Biological Markers, 7:183-188, 1992; Ledermann J A, Br. J. Cancer, 58:654, 1988; Ledermann J A, Br. J. Cancer, 68:69-73, 1993; Pedley R B, et al., Br. J. Cancer, 68:69-73, 1993; Boxer G M, et al., Br. J. Cancer, 65:825-831, 1992). Chester et al. have isolated a single chain anti-CEA antibody from a phage display library to be used in radioimmunodetection and radioimmunotherapy (U.S. Pat. No. 5,876,691), and the antibody was subsequently humanized (U.S. Pat. No. 7,232,888). Anti-CEA antibodies have also been isolated from human phage display libraries (U.S. Pat. No. 5,872,215).
The mouse monoclonal antibody PR1A3 was raised by fusion of NS1 (P3/NS1/I-Ag-4-1) myeloma cells with spleen cells from mice immunized with normal colorectal epithelium (Richman P. I. and Bodmer W. F., Int. J. Cancer, 39:317-328, 1987). PR1A3 reacts strongly to both well- and poorly-differentiated colorectal carcinomas and has advantages over other colorectal epithelium-reactive antibodies since its antigen appears to be fixed to the tumor and does not appear in the lymphatics or normal lymph nodes draining a tumor (Granowska M. et al., Eur. J. Nucl. Med., 20:690-698, 1989). For example, PR1A3 reacted with 59/60 colorectal tumors (Richman P. I. and Bodmer W. F., Int. J. Cancer, 39:317-328, 1987), whereas the CEA reactive antibody B72.3 reacted with only 75% of colorectal tumors (Mansi L., et al., Int J Rad Appl Instrum B., 16(2):127-35, 1989).
Epitope mapping of PR1A3 shows that the antibody targets the B3 domain and the GPI anchor of the CEA molecule (Durbin H. et al., Proc. Natl. Scad. Sci. USA, 91:4313-4317, 1994). Consequently, the PR1A3 antibody binds only to the membrane-bound CEA, and not the soluble CEA form that can be found in the bloodstreams of cancer patients. Because of this binding property, the PR1A3 antibody is unlikely to be sequestered by the serum CEA; instead, it can target CEA expressed on cancerous cells. The epitope bound by PR1A3 is a conformational epitope, not a linear epitope, which is thought to contribute to the loss of binding of PR1A3 to soluble CEA (Stewart et al., Cancer Immunol Immunother, 47:299-06, 1999).
The PR1A3 antibody was previously humanized by grafting the CDRs of the murine parent antibody to the heavy chain framework regions 1-3 of the human antibody RF-TS3′CL (retaining the murine framework 4 of PR1A3) and the light chain framework regions of the REI antibody. (Stewart et al., Cancer Immunol Immunother, 47:299-06, 1999). This humanized version of PR1A3 retained specificity and for surface-expressed CEA with an affinity similar to that of the murine antibody (Stewart et al., Cancer Immunol Immunother, 47:299-06, 1999; U.S. Pat. No. 5,965,710). A humanized PR1A3 (hPR1A3) antibody was shown to induce targeted killing of colorectal cancer cell lines. (Conaghhan P. J., et al., Br. J. Cancer, 98(7):1217-1225). However, the affinity of hPR1A3 for CEA is relatively low.
Radio-labeled anti-CEA antibodies have been used in clinical trials in patients with colorectal cancer. For example, an 123I-labeled chimeric minibody T84.66 (cT84.66) was used in a pilot clinical study in patients with colorectal cancer. The radio-labeled minibody was able to target cancer cells. (Wong J. Y. et al., Clin Cancer Res. 10(15):5014-21, (2004)). In another example, (131)I-labetuzumab, a radio-labeled humanized anti-CEA antibody, was tested in adjuvant radioimmunotherapy in patients with liver metastases of colorectal cancer, and was found to provide a promising survival advantage. (Liersch T., et al., Ann. Surg. Oncol. 14(9):2577-90, (2007)).
Antibody Glycosylation
The oligosaccharide component can significantly affect properties relevant to the efficacy of a therapeutic glycoprotein, including physical stability, resistance to protease attack, interactions with the immune system, pharmacokinetics, and specific biological activity. Such properties may depend not only on the presence or absence, but also on the specific structures, of oligosaccharides. Some generalizations between oligosaccharide structure and glycoprotein function can be made. For example, certain oligosaccharide structures mediate rapid clearance of the glycoprotein from the bloodstream through interactions with specific carbohydrate binding proteins, while others can be bound by antibodies and trigger undesired immune reactions. (Jenkins et al., Nature Biotechnol. 14:975-81, 1996).
Mammalian cells have been the preferred hosts for production of therapeutic glycoproteins due to their capability to glycosylate proteins in the most compatible form for human application. (Cumming et al., Glycobiology 1:115-30, 1991; Jenkins et al., Nature Biotechnol. 14:975-981, 1996). Bacteria very rarely glycosylate proteins and, like other types of common hosts, such as yeasts, filamentous fungi, insect and plant cells, yield glycosylation patterns associated with rapid clearance from the blood stream, undesirable immune interactions, and in some specific cases, reduced biological activity. Among mammalian cells, Chinese hamster ovary (CHO) cells have been most commonly used during the last two decades. In addition to giving suitable glycosylation patterns, these cells allow consistent generation of genetically stable, highly productive clonal cell lines. They can be cultured to high densities in simple bioreactors using serum-free media, and permit the development of safe and reproducible bioprocesses. Other commonly used animal cells include baby hamster kidney (BHK) cells, NS0- and SP2/0-mouse myeloma cells. More recently, production from transgenic animals has also been tested (Jenkins et al., Nature Biotechnol. 14:975-81, 1996).
All antibodies contain carbohydrate structures at conserved positions in the heavy chain constant regions, with each isotype possessing a distinct array of N-linked carbohydrate structures, which variably affect protein assembly, secretion or functional activity. (Wright A. and Morrison S. L., Trends Biotech. 15:26-32, 1997). The structure of the attached N-linked carbohydrate varies considerably, depending on the degree of processing, and can include high-mannose, multiply-branched as well as biantennary complex oligosaccharides. (Wright, A., and Morrison, S. L., Trends Biotech. 15:26-32, 1997). Typically, there is heterogeneous processing of the core oligosaccharide structures attached at a particular glycosylation site such that even monoclonal antibodies exist as a population of multiple glycoforms. Likewise, it has been shown that major differences in antibody glycosylation occur between cell lines, and even minor differences are seen for a given cell line grown under different culture conditions. (Lifely, M. R. et al., Glycobiology 5(8):813-22, 1995).
One way to obtain large increases in potency, while maintaining a simple production process and potentially avoiding significant, undesirable side effects, is to enhance the natural, cell-mediated effector functions of monoclonal antibodies by engineering their oligosaccharide component as described in Umañ a, P. et al., Nature Biotechnol. 17:176-180 (1999) and U.S. Pat. No. 6,602,684, the entire contents of which are hereby incorporated by reference in their entirety. IgG1-type antibodies, the most commonly used antibodies in cancer immunotherapy, are glycoproteins that have a conserved N-linked glycosylation site at Asn297 in each CH2 domain. The two complex biantennary oligosaccharides attached to Asn297 are buried between the CH2 domains, forming extensive contacts with the polypeptide backbone, and their presence is essential for the antibody to mediate effector functions such as antibody dependent cellular cytotoxicity (ADCC) (Lifely, M. R., et al., Glycobiology 5:813-822 (1995); Jefferis, R., et al., Immunol Rev. 163:59-76 (1998); Wright, A. and Morrison, S. L., Trends Biotechnol. 15:26-32 (1997)).
Umañ a et al. showed previously that overexpression of β(1,4)-N-acetylglucosaminyltransferase III (“GnTIII”), a glycosyltransferase catalyzing the formation of bisected oligosaccharides, in Chinese hamster ovary (CHO) cells significantly increases the in vitro ADCC activity of an anti-neuroblastoma chimeric monoclonal antibody (chCE7) produced by the engineered CHO cells. (See Umañ a, P. et al., Nature Biotechnol. 17:176-180 (1999); and International Publication No. WO 99/54342, the entire contents of which are hereby incorporated by reference). The antibody chCE7 belongs to a large class of unconjugated mAbs which have high tumor affinity and specificity, but have too little potency to be clinically useful when produced in standard industrial cell lines lacking the GnTIII enzyme (Umana, P., et al., Nature Biotechnol. 17:176-180 (1999)). That study was the first to show that large increases of ADCC activity could be obtained by engineering the antibody-producing cells to express GnTIII, which also led to an increase in the proportion of constant region (Fc)-associated, bisected oligosaccharides, including bisected, nonfucosylated oligosaccharides, above the levels found in naturally-occurring antibodies.
There remains a need for enhanced therapeutic approaches targeting CEA, in particular, membrane-bound CEA for the treatment of cancers in primates, including, but not limited to, humans.